GSM6767261: E15.5,Cholangiocyte,RNAseq,1; Mus musculus; RNA-Seq
For CUT&RUN ChIP-seq, we collected approximately 2,000–4,000 cells for each ChIP-seq, and used antibodies against H3K4me3 (Abcam, ab8580, 1ng), H3K27me3 (Millipore, 07-449, 1ng), H3K4me1 (Millipore, 07-436, 1ng), H3K27ac (Active motif, 39133, 1ng) and EZH2 (Cell Signaling Technology, 5246, 1:100). CUT&RUN ChIP-seq was performed following the protocol of Peter J Skene et al. (2018). For bulk cell RNA-seq, livers were dissociated into single cells by treatment with 0.25% trypsin at 37°C. The reaction was terminated by the addition of 0.4 volumes of FBS. The single-cell suspensions were centrifuged at 100 × g for 5 min at 4 °C, and the resulting pellets were re-suspended with PBS containing 1% FBS. Cells were incubated with the DLK-FITC antibody (MBL, D187-4, 1:100) at 4 °C for 20 min. After washing, cells were sorted with a BD FACS Aria SORP cell sorter (BD Bioscience). Dead cells were excluded by DAPI (Sigma, D9564, 0.5 µg/mL). Total RNA was extracted from approximately 50,000 cells using the RNeasy Micro Kit (Qiagen, 74004). For CUT&RUN ChIP, libraries of ChIP DNA were constructed with the Ultra DNA Library Prep Kit for Illumina (New England Biolabs, E7370) or the KAPA Hyper Prep Kit (Kapa Biosystems, KK8502). DNA purification was conducted with VAHTS DNA Clean Beads XP beads (Vazyme, N411-03). The quality of the libraries was assessed with an Advanced Analytical Fragment Analyzer (AATI). For bulk cell RNA-seq, livers were dissociated into single cells by treatment with 0.25% trypsin at 37°C. The reaction was terminated by the addition of 0.4 volumes of FBS. The single-cell suspensions were centrifuged at 100 × g for 5 min at 4 °C, and the resulting pellets were re-suspended with PBS containing 1% FBS. Cells were incubated with the DLK-FITC antibody (MBL, D187-4, 1:100) at 4 °C for 20 min. After washing, cells were sorted with a BD FACS Aria SORP cell sorter (BD Bioscience). Dead cells were excluded by DAPI (Sigma, D9564, 0.5 µg/mL). Total RNA was extracted from approximately 50,000 cells using the RNeasy Micro Kit (Qiagen, 74004). For bulk cell RNA-seq, libraries were constructed with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, #E7490) and the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs, #E7420).